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Nuclear egress of progeny capsids. Packaging of viral genomes inside capsids causes a conformational change exposing the VP2 N‐terminal on the capsid surface. (a) MVM capsids are actively exported out of the nucleus through NPCs mediated by the interaction between NS2 NES with CRM1 (Bodendorf et al., 1999; Eichwald et al., 2002; Engelsma et al., 2008; Fornerod et al., 1997; Maroto et al., 2004; Miller & Pintel, 2002). (b) The phosphorylation of the exposed VP2 N‐terminal end on the capsid surface acts as a nuclear export signal enhancing capsid export out of the nucleus (Maroto et al., 2004). (c) Phosphorylation of the capsid surface enhances capsid export (Wolfisberg et al., 2016). (d) Activation of apoptosis and necrosis affect the structure of the nuclear lamina, and capsids are released to the cytoplasm in late infection (Chen & Qiu, 2010; Nykky et al., 2010, (Wolfisberg et al., 2016). Figures created with BioRender.
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Parvoviruses are small non‐enveloped single‐stranded DNA viruses, which depend on host cell nuclear transcriptional and replication machinery. After endosomal exposure of nuclear localization sequence and a phospholipase A2 domain on the capsid surface, and escape into the cytosol, parvovirus capsids enter the nucleus. Due to the small capsid diame...
Citations
... La proteína NS2 complementa la función apoptótica de NS1. Este efecto se desarrolla a través de distintas vías en función del virus y de la célula hospedadora (Mattola et al. 2022). ...
Los parvovirus pertenecen a un grupo de virus no envueltos, capaces de infectar en una amplia variedad de hospedadores domésticos y silvestres, pudiendo causar diversos cuadros clínicos como fallas reproductivas en cerdos y bovinos, enteritis en caninos y aves de corral, panleucopenia en felinos, hepatitis en equinos, enfermedad respiratoria y cutánea en humanos. Estos virus son mayormente especie-específicos, aunque hay evidencia de transmisión interespecie, especialmente en animales silvestres. Su genoma está compuesto por una cadena lineal de ADN, de aproximadamente 5 kb, cuyas secuencias terminales son complejos palindrómicos en forma de horquilla, compuestos por 120 a 200 bases. Se encuentran ampliamente distribuidos y son muy estables en diversas condiciones ambientales, capaces de permanecer infectivos durante largos períodos. Los hospedadores susceptibles se infectan por contacto directo con individuos infectados o fómites y la infección puede ocasionar cuadros clínicos con signos diversos según la especie afectada. Además, algunos hospedadores desarrollan cuadros subclínicos que pueden eliminar el virus en secreciones y excreciones. Para su diagnóstico se emplean técnicas serológicas y moleculares, siendo la PCR la de mayor sensibilidad y especificidad. El tratamiento para los cuadros causados en animales de compañía se basa en la reversión de los signos mediante fluidoterapia, el uso de antimicrobianos de amplio espectro, antieméticos, antiácidos y protectores de la mucosa gástrica. Además, se han comenzado a utilizar antivirales, inmunomoduladores y probióticos para revertir el cuadro clínico. Por otra parte, no existe tratamiento para los cuadros clínicos en animales de producción. En producción porcina el manejo se basa en la prevención a través de la utilización de vacunas inactivadas y medidas de bioseguridad. En la presente revisión se describirán los diversos cuadros clínicos asociados a parvovirus en especies hospedadoras de interés en medicina veterinaria, y aspectos referentes a su clasificación taxonómica, epidemiología, patogenia, diagnóstico tratamiento y prevención.
... Parvoviruses enter cells by receptor-mediated endocytosis, which includes the binding of virions to specific receptors on the cell surface, followed by internalization of the virions into the host cell (9,13). However, the HBoV1 receptors remain unknown. ...
Human bocavirus 1 (HBoV1) has appeared as an emerging pathogen, causing mild to life-threatening respiratory tract infections, acute otitis media, and encephalitis in young children and immunocompromised individuals. The lack of cell lines suitable for culturing replicative viruses hinders research on HBoV1. Here, we characterized the susceptibility to HBoV1 of 29 human and 7 animal cell lines, and identified a permissive cell line, MA104. The complete HBoV1 life cycle was achieved in MA104 cells, including viral entry, complete replication, and infectious progeny virion production. Additionally, the suppression of the interferon pathway facilitated the viral genome replication in MA104 cells. RNA-sequencing showed that innate immunity, inflammation, the PI3K-Akt and MAPK signaling pathways, and the cellular membrane system were mobilized in response to HBoV1 infection. Overall, our study is the first to identify a cell line, MA104, that supports the complete HBoV1 life cycle, which will promote research on HBoV1 virology and pathogenesis and benefit drug and vaccine development.
IMPORTANCE
HBoV1 is an emerging pathogen that mainly causes respiratory tract infections, while the lack of cell lines suitable for culture replicative viruses hindered research on HBoV1. Here, we identify a permissive cell line for HBoV1 infection, MA104, and reveal that the complete life cycle of HBoV1 was supported in MA104 cells. Our findings provide a suitable cell model for the study of HBoV1 and explore its application for antiviral drug evaluation, which is vital for research on HBoV1 virology and pathogenesis, as well as for drug and vaccine development.
... Their upregulation provides additional evidence supporting the facilitation of nuclear import of AAV particles or viral components, potentially impacting viral replication or gene expression. [20][21][22][23][24] Additionally, several other biosynthetic pathways were upregulated, such as oligosaccharide-lipid intermediate processes (e.g., ALG12, MPDU1), cellular lipid biosynthesis, and glycerol ether biosynthesis (e.g., FAR1, ERLIN1) at 24 hpt. In parallel, downregulated GO biological processes included DNA-templated transcription (e.g., MAZ, SSU72), autophagy (e.g., MTMR9, FOXK1, ATP6V1C1), cellular localization, and ubiquitin protein-dependent catabolic processes (e.g., UBE2C, RNF25, FBXO7). ...
... Protein-protein interaction network reveals role of HEK293T proteins in AAV replication, assembly, and protein synthesis Our investigation showed dynamic regulation of the host proteome, which plays a pivotal role in governing the processes of AAV2 replication, assembly, and protein expression. From an extensive review of existing literature [20][21][22][23]26 and the VirHostNet 2.0 database, 24,27 we identified a subset of 124 differentially expressed host proteins associated with virus-host interactions in optimal transfectants compared with standard transfectants in at least one post-transfection time point assessed (Table S4). ...
The gene therapy field seeks cost-effective, large-scale production of recombinant adeno-associated virus (rAAV) vectors for high-dosage therapeutic applications. Although strategies like suspension cell culture and transfection optimization have shown moderate success, challenges persist for large-scale applications. To unravel molecular and cellular mechanisms influencing rAAV production, we conducted an SWATH-MS proteomic analysis of HEK293T cells transfected using standard, sub-optimal, and optimal conditions. Gene Ontology and pathway analysis revealed significant protein expression variations, particularly in processes related to cellular homeostasis, metabolic regulation, vesicular transport, ribosomal biogenesis, and cellular proliferation under optimal transfection conditions. This resulted in a 50% increase in rAAV titer compared with the standard protocol. Additionally, we identified modifications in host cell proteins crucial for AAV mRNA stability and gene translation, particularly regarding AAV capsid transcripts under optimal transfection conditions. Our study identified 124 host proteins associated with AAV replication and assembly, each exhibiting distinct expression pattern throughout rAAV production stages in optimal transfection condition. This investigation sheds light on the cellular mechanisms involved in rAAV production in HEK293T cells and proposes promising avenues for further enhancing rAAV titer during production.
... According to the current model of AAV transduction, capsids escape into the cytosol after endosomal uptake and become transported into the nucleus as intact capsids. [6][7][8] It is within the nucleus that the ssDNA becomes accessible for further processing. 9 An important step in the uncoating of AAVs is that the extended N-termini of VP1 and VP2 that normally reside within the capsid, emerge outward just before endosomal escape, in a likely pH-triggered event. ...
... [10][11][12][13] This initial step then uncovers different nuclear localization sequences which are vital for nuclear uptake of AAVs either by the nuclear pore complex or via pore formation in the nuclear envelope. 6,8 Once inside the nucleus, however, the process of AAV uncoating and ssDNA release remains elusive. ...
Adeno-associated viruses (AAVs) are gaining traction as delivery vehicles for gene therapy although the molecular understanding of AAV-transgene release is still limited. Typically, the process of viral uncoating is investigated ( in vitro ) through thermal stress, revealing capsid disintegration at elevated temperatures. Here, we used single-molecule interferometric scattering microscopy to assess the (in)stability of different empty and filled AAV preparations. By introducing a heat-stable DNA plasmid as an internal standard, we quantitatively probed the impact of heat on AAVs. Generally, empty AAVs exhibited greater heat resistance than genome-filled particles. Our data also indicate that upon DNA release, the capsids do not transform into empty AAVs, but seem to aggregate or disintegrate. Strikingly, some AAVs exhibited an intermediate state with disrupted capsids but preserved bound genome, a feature that experimentally only emerged following incubation with a nuclease. Our data demonstrate that the thermal uncoating process is highly AAV specific ( i . e ., can be influenced by serotype, genome, host system). We argue that nuclease treatment in combination with mass photometry can be used as an additional analytical tool for assessing structural integrity of recombinant and/or clinical AAV vectors.
... A long-standing unanswered topic is how and where the capsid uncoating for gene expression before entering the nucleus (42,43). Earlier studies have shown that in the infectious parvovirus particles, the capsid surface exposed the 5' end 20-30 nucleotides (44)(45)(46)(47). ...
Parvoviruses are a group of non-enveloped DNA viruses that have a broad spectrum of natural infections, making them important in public health. NS1 is the largest and most complex non-structural protein in the parvovirus genome, which is indispensable in the life cycle of parvovirus and is closely related to viral replication, induction of host cell apoptosis, cycle arrest, DNA damage response (DDR), and other processes. Parvovirus activates and utilizes the DDR pathway to promote viral replication through NS1, thereby increasing pathogenicity to the host cells. Here, we review the latest progress of parvovirus in regulating host cell DDR during the parvovirus lifecycle and discuss the potential of cellular consequences of regulating the DDR pathway, targeting to provide the theoretical basis for further elucidation of the pathogenesis of parvovirus and development of new antiviral drugs.
... Their upregulation provides additional evidence supporting the facilitation of nuclear import of AAV particles or viral components, potentially impacting viral replication or gene expression. [20][21][22][23][24] Additionally, several other biosynthetic pathways were upregulated, such as oligosaccharide-lipid intermediate processes (e.g., ALG12, MPDU1), cellular lipid biosynthesis, and glycerol ether biosynthesis (e.g., FAR1, ERLIN1) at 24 hpt. In parallel, downregulated GO biological processes included DNA-templated transcription (e.g., MAZ, SSU72), autophagy (e.g., MTMR9, FOXK1, ATP6V1C1), cellular localization, and ubiquitin protein-dependent catabolic processes (e.g., UBE2C, RNF25, FBXO7). ...
... Protein-protein interaction network reveals role of HEK293T proteins in AAV replication, assembly, and protein synthesis Our investigation showed dynamic regulation of the host proteome, which plays a pivotal role in governing the processes of AAV2 replication, assembly, and protein expression. From an extensive review of existing literature [20][21][22][23]26 and the VirHostNet 2.0 database, 24,27 we identified a subset of 124 differentially expressed host proteins associated with virus-host interactions in optimal transfectants compared with standard transfectants in at least one post-transfection time point assessed (Table S4). ...
The gene therapy field is actively pursuing cost-effective, large-scale production of recombinant adeno-associated virus (rAAV) vectors for therapeutic applications, which demand high dosages. Enhanced yield is essential but presents technical and cost challenges. Strategies like suspension cell culture, transfection optimization, and cultivation conditions have shown moderate success but fall short for large-scale applications. In this study, we explore the host cell proteome of HEK293T cell lines used for AAV2 production under different transfection conditions (standard, sub-optimal, optimal) using SWATH-MS. To understand molecular and cellular mechanisms, we created a tailored spectral library for HEK293T-AAV interactions. Our gene ontology and pathway analysis revealed significant protein expression variations, particularly in processes related to cellular homeostasis, metabolic regulation, vesicular transport, ribosomal biogenesis, and cellular proliferation under optimal transfection conditions. These conditions increased rAAV titre by 50% compared to standard protocols. Furthermore, we identified alterations in host cell proteins crucial for AAV mRNA stability and gene translation, particularly regarding AAV capsid transcripts in optimal transfection conditions. This study provides insights into cellular mechanisms during rAAV production in HEK293T cells and offers potential advancements in scalability and cost-efficiency for gene therapy vector production.
Significance
Generating AAV is challenging and the amount produced is limited, despite the success of gene therapy. Thus, it is necessary to enhance the productivity of host cells through various engineering techniques. The replication and transduction of viruses cause numerous modifications to the host cell’s proteome. Viruses exploit the processes of the host cell, influencing the availability, post-translational adjustments, associations, or placement of its proteins during the replication/growth cycle. Comprehending the fluctuations in biological processes during AAV replication is essential to grasping how virus-host relations shape the result of infection and viral protein expression.
... NP1 processes viral pre-mRNA. NP1 is needed to define the big 3′ exon, the VP-encoding exon (Shao et al., 2021;Mattola et al., 2022). ...
... The efficient assembly of these VLPs is essential for the virus's ability to establish and maintain an infection. Understanding the role of HBoV-1's capsid proteins and their interactions with host cells is crucial for developing effective antiviral therapies and vaccines (Kailasan et al., 2016;Liu et al., 2017;Ros et al., 2017;Malta et al., 2020;Fakhiri and Grimm, 2021;Shao et al., 2021;Mattola et al., 2022;Xie et al., 2022). ...
... Understanding the structural and functional differences between different strains of HBoV is critical for developing effective strategies for diagnosing and treating viral infections. By examining how these viruses interact with host cells and evade the immune system, researchers can gain insight into the underlying mechanisms of viral infection and identify new targets for drug and vaccine development (Babkin et al., 2013;Abramczuk et al., 2015;Liu et al., 2017;Qiu et al., 2017;Ros et al., 2017;Christensen et al., 2019;Malta et al., 2020;Bhat and Almajhdi, 2021;Shao et al., 2021;Mattola et al., 2022;Xie et al., 2022). ...
The single-stranded DNA virus known as human bocavirus 1 (HBoV-1) is an icosahedral, linear member of the Parvoviridae family. In 2005, it was discovered in nasopharyngeal samples taken from kids who had respiratory tract illnesses. The HBoV genome is 4.7–5.7 kb in total length. The HBoV genome comprises three open-reading frames (ORF1, ORF2, and ORF3) that express structural proteins (VP1, VP2, and VP3), viral non-coding RNA, and non-structural proteins (NS1, NS1-70, NS2, NS3, and NP1) (BocaSR). The NS1 and NP1 are crucial for viral DNA replication and are substantially conserved proteins. Replication of the HBoV-1 genome in non-dividing, polarized airway epithelial cells. In vitro, HBoV-1 infects human airway epithelial cells that are strongly differentiated or polarized. Young children who have HBoV-1 are at risk for developing a wide range of respiratory illnesses, such as the common cold, acute otitis media, pneumonia, and bronchiolitis. The most common clinical symptoms are wheezing, coughing, dyspnea, and rhinorrhea. After infection, HBoV-1 DNA can continue to be present in airway secretions for months. The prevalence of coinfections is considerable, and the clinical symptoms can be more severe than those linked to mono-infections. HBoV-1 is frequently detected in combination with other pathogens in various reports. The fecal-oral and respiratory pathways are more likely to be used for HBoV-1 transmission. HBoV-1 is endemic; it tends to peak in the winter and spring. This Review summarizes the knowledge on HBoV-1.
... In addition, MVM can be internalized i and contains signals that modulate its intracellular trafficking. Durin pores in its capsid allow both the internalization and externalization o DNA genome and of peptide signals (reviewed in [39,40]). ...
... In addition, MVM can be internalized in different cell types and contains signals that modulate its intracellular trafficking. During infection by MVM, pores in its capsid allow both the internalization and externalization of the single-stranded DNA genome and of peptide signals (reviewed in [39,40]). ...
Citation: Fuertes, M.A.; López Mateos, D.; Valiente, L.; Rodríguez Huete, A.; Valbuena, A.; Mateu, M.G. Electrostatic Screening, Acidic pH and Macromolecular Crowding Increase the Self-Assembly Efficiency of the Minute Virus of Mice Capsid In Vitro. Viruses 2023, 15, 1054. https://
... In addition, MVM can be internalized i and contains signals that modulate its intracellular trafficking. Durin pores in its capsid allow both the internalization and externalization o DNA genome and of peptide signals (reviewed in [39,40]). ...
... In addition, MVM can be internalized in different cell types and contains signals that modulate its intracellular trafficking. During infection by MVM, pores in its capsid allow both the internalization and externalization of the single-stranded DNA genome and of peptide signals (reviewed in [39,40]). ...
The hollow protein capsids from a number of different viruses are being considered for multiple biomedical or nanotechnological applications. In order to improve the applied potential of a given viral capsid as a nanocarrier or nanocontainer, specific conditions must be found for achieving its faithful and efficient assembly in vitro. The small size, adequate physical properties and specialized biological functions of the capsids of parvoviruses such as the minute virus of mice (MVM) make them excellent choices as nanocarriers and nanocontainers. In this study we analyzed the effects of protein concentration, macromolecular crowding, temperature, pH, ionic strength, or a combination of some of those variables on the fidelity and efficiency of self-assembly of the MVM capsid in vitro. The results revealed that the in vitro reassembly of the MVM capsid is an efficient and faithful process. Under some conditions, up to ~40% of the starting virus capsids were reassembled in vitro as free, non aggregated, correctly assembled particles. These results open up the possibility of encapsidating different compounds in VP2-only capsids of MVM during its reassembly in vitro, and encourage the use of virus-like particles of MVM as nanocontainers.
... Recently, a novel paradigm proposed that importin-beta (impβ) alone mediates viral capsid passage through the nuclear envelope [16]. In 2022 Mattola et al. at the University of Jyväskylä, Finland discovered that parvovirus nucleation follows a new pattern [17]. This pattern involves the temporary dissolution of the nuclear envelope while Imp acts, allowing the genome to pass through. ...
A group of DNA viruses called parvoviruses that have significant effects on cancer therapy and genetic engineering applications. After passing through the cell membrane to reach the cytosol, it moves along the microtubule toward the nuclear membrane. The nuclear localization signal (NLS) is recognized by importin-beta (impβ) and other proteins from the complex outside the nuclear membrane and binds to enter the nucleus via the nuclear pore complex (NPC). There are two main pathways for viruses to enter the nucleus. The classical pathway is through the interaction of imp α and impβ with NLS via NPC. The other is the NPC mediated by the combination of impβ and it. While the capsid is introduced into the nucleus through classical nuclear transduction, there is also a transient nuclear membrane dissolution leading to passive transport into the nucleus, which has been proposed in recent years. This article mainly discusses several nuclear entry pathways and related proteins, providing a reference for subsequent research on viral entry pathways.
